Virulent strains of the soil bacterium Agrobacterium tumefaciens are known to infect dicotyledonous plants and to elicit a neoplastic response in these plants. The tumor-inducing agent in the bacterium is a plasmid that functions by transferring some of its DNA into its host plant's cells where it is integrated into the chromosomes of the host plant's cells. This plasmid is called the Ti plasmid, and the virulence of the various strains of A. tumefaciens is determined in part by the vir region of the Ti plasmid which is responsible for mobilization and transfer of the T-DNA. The T-DNA section is delimited by two 23-base-pair repeats designated right border and left border, respectively. Any genetic information placed between these two border sequences may be mobilized and delivered to a susceptible host. Once incorporated into a chromosome, the T-DNA genes behave like normal dominant plant genes. They are stably maintained, expressed and sexually transmitted by transformed plants, and they are inherited in normal Mendelian fashion.
The lump of plant tumor tissue that grows in an undifferentiated way at the site of the A. tumefaciens infection is called a crown gall. Cells of crown gall tumors induced by A. tumefaciens synthesize unusual amino acids called opines. Different strains of A. tumefaciens direct the synthesis of different opines by the crown gall cells, and the particular opine induced is a characteristic of the strain which infected the plant. Further, the ability to catabolize the particular opine induced by a given strain is also characteristic of that strain.
Opines are not normally synthesized by A. tumefaciens or by the uninfected host plants. Although it is the T-DNA which codes for the enzymes involved in the synthesis of the opines, the opine synthases, these genes are expressed only in infected plant tissue. This type of expression is consistent with the observation that these genes are under the control of eukaryotic regulatory sequences on the T-DNA.
The most common opines are octopine and nopaline. The opine synthase that catalyzes the synthesis of octopine is lysopine dehydrogenase, and the opine synthase that catalyzes the synthesis of nopaline is nopaline dehydrogenase.
When crown gall cells are put into culture, they grow to form a callus culture even in media devoid of the plant hormones that must be added to induce normal plant cells to grow in culture. A callus culture is a disorganized mass of relatively undifferentiated plant cells. This ability of crown gall cells to grow in hormone-free media is also attributable to the presence of the T-DNA in the transformed host plant cells since genes which direct the synthesis of phyto-hormone are also associated with the T-DNA.
A DNA segment foreign to the A. tumefaciens and to the host plant which is inserted into the T-DNA by genetic manipulation will also be transferred to host plant's cells by A. tumefaciens. Thus, the Ti plasmid can be used as a vector for the genetic engineering of host plants. Although, in wild type A. tumefaciens there is only one Ti plasmid per bacterium, in genetically-engineered A. tumefaciens, the vir region and the T-DNA do not have to be carried on the same Ti plasmid for transfer of the T-DNA to occur. The vir region and the T-DNA can be carried on separate plasmids contained within the same Agrobacterium.
It has generally been assumed that the host range of A. tumefaciens was limited to the dicotyledons, and that transformation of monocotyledons could not be effected with this bacterium. Indeed, no one has reported the transformation of any member of the monocotyledonous Gramineae family by infection with A. tumefaciens.
However, recently, Hooykaas-Van Slogteren et al., in Nature, 311, 763 (1984), reported the production of small swellings at wound sites infected with A. tumefaciens on monocotyledons of the Liliaceae and Amaryllidaceae families. Opines were detected in plant cells taken from the wound sites of the infected plants.
Also, Hernalsteens et al. reported in The EMBO Journal, 3, 3039 (1984) that cultured stem fragments of the monocotyledon Asparagus officinalis, a member of the family Liliaceae, infected with A. tumefaciens strain C58 developed tumorous proliferations. One of these tumorous proliferations could be propagated on hormone-free medium, and opines were detected in the established callus culture derived from this tumorous proliferation.
In 1982, Anne C. F. Graves reported in her Ph.D. Dissertation entitled "Some Tumorigenic Activities of Agrobacterium Tumefaciens (Smith and Town) Conn." (Bowling Green State University) that irregular masses of tissue developed on gladiolus discs in response to inoculations with A. tumefaciens C58N and B6. These masses of tissue appeared to be the same as, and have cellular morphology similar to, those that develop on potato tuber discs. Gladiolus is a member of the monocotyledonous Iridaceae family. A compound that co-migrated with the octopine standard during electrophoresis was found in the proliferations on the gladiolus discs that were induced by strain B6, and one that migrated just behind the octopine standard occurred in those induced by C58N. Also, octopine dehydrogenase was found in extracts of the cellular proliferations induced by A. tumefaciens B6 but not in those induced by A. tumefaciens C58N.
Dr. Graves also described the response of certain other monocots to inoculation with A. tumefaciens. No cellular proliferation was observed on ginger root rhizome discs, and the results with tulip bulb discs were inconclusive. Cellular proliferations on discs of the rhizomes of cattail and skunk cabbage were limited to several layers of clear cells at the ends of vascular bundles in the early spring.
DeCleene and DeLey in The Botanical Review, 42, 389 (1976) reported the results of an extensive study of the plant host range of A. tumefaciens. Their article teaches that monocots of the orders Liliales and Arales are susceptible to infection with A. tumefaciens but that monocotyledons in general are unsusceptible to A. tumefaciens infection. In particular, their article reports that the Gramineae tested were not susceptible to infection with A. tumefaciens. Susceptibility to A. tumefaciens infection was determined by whether a swelling or tumor developed at the wound site.
Lorz et al. in Mol. Gen. Genet., 199, 178 (1985), Fromm et al in Nature, 319, 791 (1986) and Portrykus et al. in Mol. Gen. Genet, 199, 183 (1985) have reported the transformation of Gramineae by direct gene transfer to protoplasts. Protoplasts are plant cells from which the cell wall has been removed by digestion with enzymes. Lorz et al. transformed protoplasts of Triticum monococcum using a DNA construct containing the nopaline synthase promotor and the polyadenylation regulatory signal of the octopine synthase gene. Fromm et al discloses that the electroporation-mediated transfer of plasmid pCaMVNEO (comprising the cauliflower mosaic virus 35 S promoter, the neomycin phosphotransferase II gene from the transposon Tn5 and the nopaline synthase 3' region) into maize protoplasts results in stably-transformed maize cells that are resistant to kanamycin.
To obtain transformed plants from the transformed cells generated using either the infection techniques of Hooykaas-Van Slogteren et al., Hernalsteens et al., Graves and DeCleene and DeLey or the direct gene transfer techniques of Lorz et al., Fromm et al. and Portrykus et al., the plants would have to be regenerated from protoplasts or single cell cultures. However, no one has yet been able to regenerate plants from protoplasts or single cell cultures of the Gramineae. Indeed, no means of producing transformed plants or other transformed differentiated organs or tissues of the Gramineae is currently known, and no method yet exists for transforming the Gramineae in a manner which allows for the expression of exogenous DNA in agriculturally important forms or parts of the Gramineae such as seeds, pollen, ears or plants.
Finally, PCT International Publication No. WO 86/00931 (Simpson et al.) published Feb. 13, 1986, teaches in vivo methods for transforming and regenerating intact plants. This patent application discloses that the methods of the invention can be used for the transformation of any plant that forms a shooty tumor following infection with an A. tumefaciens shooty mutant strain. However, as noted above, the Gramineae are not known to produce tumors or even swellings in response to inoculation with A. tumefaciens. In the practice of the present invention, no tumors, swellings or cellular proliferations of any kind have been observed on the Gramineae in response to inoculation with A. tumefaciens.